This application is in response to NOT-OD-09-058, "NIH announces the availability of Recovery Act funds for competitive revision applications". My R01 investigates the biochemical and cellular functions of mammalian formin proteins, which are potent actin assembly factors. Mammals possess 15 distinct formins, and cellular function is poorly understood for many of these isoforms. I focus on two mammalian formins, INF2 and FRL2. INF2 is unique in two respects. Biochemically, INF2 accelerates both actin polymerization and depolymerization. In cells, INF2 localizes to ER membranes. INF2 also binds microtubules (MTs) tightly. FRL2 bundles actin filaments in vitro and causes robust filopodia assembly in cells. The aims of the original application were to: 1) dissect motifs in INF2 and FRL2 mediating effects on actin/MTs;2) determine regulatory mechanisms for INF2 and FRL2;and 3) analyze the cellular roles of INF2 and FRL2. I proposed to conduct the first two aims using conventional population-based biochemical techniques. I proposed to conduct live cell microscopy in aim 3 in collaboration with an expert in the field. This competitive revision expands these aims by addressing mechanism at a much more detailed level. This expansion is made possible by investments in two areas: 1) equipment purchases that dramatically increase our analytical capability;and 2) addition of personnel. The requested equipment will up- grade my fluorescence microscope by: 1) allowing Total Internal Reflection (TIRF) microscopy;and 2) allowing near simultaneous red/green image acquisition in live cells. For personnel, I propose to add one graduate student. Expansion of aims will be as follows. In Aim 1, we will dissect the mechanism of INF2's depolymerization activity, as well as its effect on MT dynamics. In Aim 2, we will establish a cell-free system for INF2- mediated actin and MT dynamics on ER membrane. In Aim 3, we will correlate FRL2 dynamics with those of actin in live cells. PUBLIC HEALTH RELEVANCE: Actin is a protein that makes up the skeletons of cells, and whose polymerization is vital for at least 15 processes in human cells, including processes involved in cell division, neural function, and immune cell function. My laboratory studies how actin polymerization is controlled in specific processes. By understanding these processes, I hope to develop therapeutic interventions for diseases such as cancer and immunological diseases.